• 文献标题:   Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide
  • 文献类型:   Article
  • 作  者:   BENJAMIN CJ, WRIGHT KJ, BOLTON SC, HYUN SH, KRYNSKI K, GROVER M, YU GM, GUO F, KINZERURSEM TL, JIANG W, THOMPSON DH
  • 作者关键词:  
  • 出版物名称:   SCIENTIFIC REPORTS
  • ISSN:   2045-2322
  • 通讯作者地址:   Purdue Univ
  • 被引频次:   6
  • DOI:   10.1038/srep32500
  • 出版年:   2016

▎ 摘  要

We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with N-alpha, N-alpha-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 angstrom. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.